The present invention relates to a novel human gene encoding a polypeptide which is a novel human cytokine. More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named Interleukin 20, hereinafter referred to as xe2x80x9cIL-20xe2x80x9d. IL-20 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to the immune system, and therapeutic methods for treating such disorders.
The invention further relates to screening methods for identifying agonists and antagonists of IL-20 activity.
Cytokines typically exert their respective biochemical and physiological effects by binding to specific receptor molecules. Receptor binding will then stimulate specific signal transduction pathways (Kishimoto, T., et al., Cell 76:253-262 (1994). The specific interactions of cytokines with their receptors are often the primary regulators of a wide variety of cellular processes including activation, proliferation, and differentiation (Arai, K.-I, et al., Ann. Rev. Biochem. 59:783-836 (1990); Paul, W. E. and Seder, R. A., Cell 76:241-251 (1994)).
Human interleukin (IL)-17 was only recently identified. IL-17 is a 155 amino acid polypetide which was molecularly cloned from a CD4+ T-cell cDNA library (Yao, Z., et al., J. Immunol. 155:5483-5486 (1995)). The IL-17 polypeptide contains an N-terminal signal peptide and contains approximately 72% identity at the amino acid level with a T-cell trophic herpesvirus saimiri (HVS) gene designated HVS13. High levels of IL-17 are secreted from CD4-positive primary peripheral blood leukocytes (PBL) upon stimulation (Yao, Z., et al., Immunity 3:811-821 (1995)). Treatment of fibroblasts with IL-17, HVS13, or another murine homologue, designated CTLA8, activate signal transduction pathways and result in the stimulation of the NF-xcexaB transcription factor family, the secretion of IL-6, and the costimulation of T-cell proliferation (Yao, Z., et al., Immunity 3:811-821 (1995)).
An HVS13-Fc fusion protein was used to isolate a murine IL-17 receptor molecule which does not appear to belong to any of the previously described cytokine receptor families (Yao, Z., et al., Immunity 3:811-821 (1995)). The murine IL-17 receptor (mIL-17R) is predicted to encode a type I transmembrane protein of 864 amino acids with an apparent molecular mass of 97.8 kDa. mIL-17R is predicted to possess an N-terminal signal peptide with a cleavage site between alanine-31 and serine-32. The molecule also contains a 291 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 521 amino acid cytoplasmic tail. A soluble recombinant IL-17R molecule consisting of 323 amino acids of the extracellular domain of IL-17R fused to the Fc portion of human immunoglobulin IgG1 was able to significantly inhibit IL-17-induced IL-6 production by murine NIH-3T3 cells (supra).
Interestingly, the expression of the IL-17 gene is highly restricted. It is typically observed primarily in activated T-lymphocyte memory cells (Broxmeyer, H. J. Exp. Med. 183:2411-2415 (1996); Fossiez, F., et al., J. Exp. Med. 183:2593-2603 (1996)). Conversely, the IL-17 receptor appears to be expressed in a large number of cells and tissues (Rouvier, E., et al., J. Immunol. 150:5445-5456 (1993); Yao, Z., et al., J. Immunol. 155:5483-5486 (1995)). It remains to be seen, however, if IL-17 itself can play an autocrine role in the expression of IL-17. IL-17 has been implicated as a causitive agent in the expression of IL-6, IL-8, G-CSF, Prostaglandin E (PGE2), and intracellular adhesion molecule (ICAM)-1 (Fossiez, F., supra; Yao, Z., et al., Immunity 3:811-821 (1995)). Each of these molecules possesses highly relevent and potentially therapeutically valuable properties. For instance, IL-6 is involved in the regulation of hematopoietic stem and progenitor cell growth and expansion (Ikebuchi, K., et al., Proc. Natl. Acad. Sci. USA 84:9035-9039 (1987); Gentile, P. and Broxmeyer, H. E. Ann. N.Y. Acad. Sci. USA 628:74-83 (1991)). IL-8 exhibits a myelosuppressive activity for stem cells and immature subsets of myeloid progenitors (Broxmeyer, H. E., et al., Ann. Hematol. 71:235-246 (1995); Daly, T. J., et al., J. Biol. Chem. 270:23282-23292 (1995)). G-CSF acts both early and late to activate and stimulate hematopoiesis in general, and more specifically on neutrophil hematopoiesis, while PGE2 enhances erythropoiesis, suppresses lymphopoiesis and myelopoiesis in general, and strongly suppresses monocytopoiesis (Broxmeyer, H. E. Amer. J. Ped. Hematol./Oncol. 14:22-30 (1992); Broxmeyer, H. E. and Williams, D. E. CRC Crit. Rev. Oncol./Hematol. 8:173-226 (1988)).
Thus, there is a need for polypeptides that function as immunoregulatory molecules and, thereby, function in the transfer of an extracelluIlar signal ultimately to the nucleus of the cell, since disturbances of such regulation may be involved in disorders relating to cellular activation, hemostasis, angiogenesis, tumor metastasis, cellular migration and ovulation, as well as neurogenesis. Therefore, there is a need for identification and characterization of such human polypeptides which can play a role in detecting, preventing, ameliorating or correcting such disorders.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the IL-20 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNA clone deposited as plasmid DNA as ATCC Deposit Number 209232 on Aug. 29, 1997. The nucleotide sequence determined by sequencing the deposited IL-20 clone, which is shown in FIG. 1 (SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 180 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 45-47, and a predicted molecular weight of about 20.4 kDa. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence excepting the N-terminal methionine shown in SEQ ID NO:2, or the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone in ATCC Deposit Number 209232, which molecules also can encode additional amino acids fused to the N-terminus of the IL-20 amino acid sequence.
The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the IL-20 polypeptide having the complete amino acid sequence shown in SEQ ID NO:15 or the complete amino acid sequence encoded by the cDNA clone deposited in a pool of 50 distinct plasmid DNA molecules as ATCC Deposit Number 209138 on Jul. 3, 1997. The sense and antisense nucleotide sequences determined by 209138 on Jul. 3, 1997. The sense and antisense nucleotide sequences determined by respectively, contain an open reading frame in the sense sequence (SEQ ID NO:28) encoding a polypeptide of 118 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 59-61. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence excepting the N-terminal methionine shown in SEQ ID NO:15, or the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone in ATCC Deposit Number 209138, which molecules also can encode additional amino acids fused to the N-terminus of the IL-20 amino acid sequence. Nucleic acid molecules of the invention further include those encoding any of the N-terminal and/or C-terminal IL-20 deletion mutations with termini of n3 and/or m3 as set forth below.
IL-20 has a predicted leader sequence of 20 amino acids underlined in FIG. 1; and the amino acid sequence of the predicted mature IL-20 protein is also shown in FIG. 1, as amino acid residues 21-180 and as residues 1-160 in SEQ ID NO:2. The encoded polypeptide also has a predicted leader sequence of 20 amino acids of the IL-20 polypeptide of the invention as shown as amino acid residues 1-20 in SEQ ID NO:15, and a predicted mature protein comprising amino acids residues 21-118 of the IL-20 polypeptide of the invention as shown in SEQ ID NO:15.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence in SEQ ID NO:2 (i.e., positions xe2x88x9220 to 160 of SEQ ID NO:2); (b) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence in SEQ ID NO:15 (i.e., positions 1 to 118 of SEQ ID NO:2); (c) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence in SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions xe2x88x9219 to 160 of SEQ ID NO:2); (d) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence in SEQ ID NO:15 excepting the N-terminal methionine (i.e., positions 2 to 118 of SEQ ID NO:15); (e) a nucleotide sequence encoding the predicted mature IL-20 polypeptide having the amino acid sequence at positions 1 to 160 in SEQ ID NO:2; (f) a nucleotide sequence encoding the predicted mature IL-20 polypeptide having the amino acid sequence at positions 21 to 118 in SEQ ID NO:15; (g) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209232; (h) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209138; (i) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone contained in ATCC Deposit No. 209232; (j) a nucleotide sequence encoding the IL-20 polypeptide having the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone contained in ATCC Deposit No. 209138; (k) a nucleotide sequence encoding the mature IL-20 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209232; (l) a nucleotide sequence encoding the mature IL-20 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209138; and, (m) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k) or (l), above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical to (that is to say, at most 10% different from), and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (that is to say, at most 5%, 4%, 3%, 2% or 1% different from), any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l) or (m), above, or a polynucleotide which hybridizes under stringent hybridization. conditions to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l) or (m), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolatled nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of an IL-20 polypeptide having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k) or (l), above. A further nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an IL-20 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably not more than 30 conservative amino acid substitutions, and still even more preferably not more than 20 conservative amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a polynucleotide which encodes the amino acid sequence of an IL-20 polypeptide to have an amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of IL-20 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated IL-20 polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the full-length IL-20 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 (i.e., positions xe2x88x9220 to 160 of SEQ ID NO:2); (b) the amino acid sequence of the full-length IL-20 polypeptide having the complete amino acid sequence shown in SEQ ID NO:15 (i.e., positions 1 to 118 of SEQ ID NO:2); (c) the amino acid sequence of the full-length IL-20 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions xe2x88x9219 to 160 of SEQ ID NO:2); (d) the amino acid sequence of the full-length IL-20 polypeptide having the complete amino acid sequence shown in SEQ ID NO:15 excepting the N-terminal methionine (i.e., positions 2 to 118 of SEQ ID NO:15); (e) the amino acid sequence of the predicted mature IL-20 polypeptide having the amino acid sequence at positions 1 to 160 in SEQ ID NO:2; (f) the amino acid sequence of the predicted mature IL-20 polypeptide having the amino acid sequence at positions 21 to 118 in SEQ ID NO:15; (f) the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 209232; (g) the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 209138; (h) the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone contained in the ATCC Deposit No. 209232; (i) the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone contained in the ATCC Deposit No. 209138; (j) the complete amino acid sequence of the predicted mature IL-20 polypeptide encoded by the cDNA clone contained in the ATCC Deposit No. 209232; and, (k) the complete amino acid sequence of the predicted mature IL-20 polypeptide encoded by the cDNA clone contained in the ATCC Deposit No. 209138. The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 80% identical to (that is to say, at most 20% different from), more preferably at least 90% identical to (that is to say, at most 10% different from), and still more preferably 95%, 96%, 97%, 98% or 99% identical to (that is to say, at most 5%, 4%, 3%, 2% or 1% different from) those described in (a), (b), (c), (d), (c), (f), (g), (h), (i), (j) or (k), above, as well as polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity, to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an epitope-bearing portion of an IL-20 polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of an IL-20 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
A further embodiment of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an IL-20 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably not more than 30 conservative amino acid substitutions, and still even more preferably not more than 20 conservative amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of an IL-20 polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5,14, 3, 2 or 1 conservative amino acid substitutions.
In another embodiment, the invention provides an isolated antibody that binds specifically to an IL-20 polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k), above. The invention further provides methods for isolating antibodies that bind specifically to an IL-20 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
The invention also provides for pharmaceutical compositions comprising IL-20 polypeptides, particularly human IL-20 polypeptides, which may be employed, for instance, to treat disorders relating to the proliferation or differentiation of T-cells, cellular activation, hemostasis, angiogenesis, tumor metastasis, cellular migration and ovulation, as well as neurogenesis. Methods of treating individuals in need of IL-20 polypeptides are also provided.
The invention further provides compositions comprising an IL-20 polynucleotide or an IL-20 polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise an IL-20 polynucleotide for expression of an IL-20 polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of IL-20.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a biological activity of the IL-20 polypeptide, which involves contacting a receptor which is enhanced by the IL-20 polypeptide with the candidate compound in the presence of an IL-20 polypeptide, assaying the IL-6 secretion or lymphocyte proliferation activity of the receptor in the presence of the candidate compound and of IL-20 polypeptide, and comparing the receptor activity to a standard level of activity, the standard being assayed when contact is made between the receptor and in the presence of the IL-20 polypeptide and the absence of the candidate compound In this assay, an increase in receptor activity over the standard indicates that the candidate compound is an agonist of IL-20 activity and a decrease in receptor activity compared to the standard indicates that the compound is an antagonist of IL-20 activity.
In another aspect, a screening assay for)agonists and antagonists is provided which involves determining the effect a candidate compound has on IL-20 binding to a receptor. In particular, the method involves contacting the receptor with an IL-20 polypeptide and a candidate compound and determining whether IL-20 polypeptide binding to the receptor is increased or decreased due to the presence of the candidate compound. In this assay, an increase in binding of IL-20 over the standard binding indicates that the candidate compound is an agonist of IL-20 binding activity and a decrease in IL-20 binding compared to the standard indicates that the compound is an antagonist of IL-20 binding activity.
In yet another aspect, the IL-20 polypeptide may bind to a cell surface protein which also function as a viral receptor or coreceptor. Thus, IL-20, or agonists or antagonists thereof, may be used to regulate viral infectivity at the level of viral binding or interaction with the IL-20 receptor or coreceptor or during the process of viral internalization or entry into the cell.
It has been discovered that IL-20 is expressed not only in thymus, but also in thymus tumor and 12 week old whole human embryo. Therefore, nucleic acids of the invention are useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to those polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). In addition, for a number of disorders of the above tissues or cells, particularly of the immune, significantly higher or lower levels of IL-20 gene expression may be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d IL-20 gene expression level, i.e., the IL-20 expression level in healthy tissue from an individual not having the immune system disorder. Thus, the invention provides a diagnostic method useful during diagnosis of such a disorder, which involves: (a) assaying IL-20 gene expression level in cells or body fluid of an individual; (b) comparing the IL-20 gene expression level with a standard IL-20 gene expression level, whereby an increase or decrease in the assayed IL-20 gene expression level compared to the standard expression level is indicative of disorder in the immune system.
A further consequence of the observed thymus-restricted expression of endogenous IL-20 is that the IL-20 of the present invention may be useful in the regulation of the proliferation or differentiation of T-cells in general, for specific subsets of T-cells, for other immune cells in general, for other specific subsets of other immune cells, or any combination thereof. Thus, IL-20 of the present invention may be used therapeutically to treat disorders related to the immune system, including autoimmune and hematopoietic diseases or disorders, including AIDS, arthritis, or normal or abnormal cellular or systemic processes related to aging, and the like.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of IL-20 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated IL-20 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of IL-20 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an IL-20 antagonist. Preferred antagonists for use in the present invention are IL-20-specific antibodies.